Optimization of experimental design parameters for high-throughput chromatin immunoprecipitation studies.
High-throughput, microarray-based chromatin immunoprecipitation (ChIP-chip) technology allows in vivo elucidation of transcriptional networks. However this complex is not yet readily accessible, in part because its many parameters have not been systematically evaluated and optimized. We address this gap by systematically assessing experimental-design parameters including antibody purity, dye-bias, array-batch, inter-day hybridization bias, amplification method and choice of hybridization control. The combined performance of these optimized parameters shows a 90% validation rate in ChIP-chip analysis of Myc genomic binding in HL60 cells using two different microarray platforms. Increased sensitivity and decreased noise in ChIP-chip assays will enable wider use of this methodology to accurately and affordably elucidate transcriptional networks.
Ponzielli
Romina
R
Division of Cancer Genomics and Proteomics, Ontario Cancer Institute, University Health Network, University of Toronto, Toronto, ON, Canada.
Boutros
Paul C
PC
Katz
Sigal
S
Stojanova
Angelina
A
Hanley
Adam P
AP
Khosravi
Fereshteh
F
Bros
Christina
C
Jurisica
Igor
I
Penn
Linda Z
LZ
D000906:Antibodies / Q000276:immunology; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D018922:HL-60 Cells; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D016271:Proto-Oncogene Proteins c-myc / Q000276:immunology / Q000378:metabolism; D050436:Regulatory Elements, Transcriptional
D000906:Antibodies; D016271:Proto-Oncogene Proteins c-myc